The invention relates to a method for infiltrating tissue samples with carrier material, said tissue sample being treated in multiple infiltration steps with carrier material of an increasing degree of purity. The invention further relates to an apparatus for carrying out the method.
Biological tissue samples, in particular histological tissue samples, are often required in the fields of human and veterinary medicine, in particular as microscopic prepared specimens for the assessment of cells and their environment. For microscopic inspection, thin sections of the tissue sample must be prepared for assessment under the microscope, in incident or transmitted light, by an expert. The tissue sample must have a certain strength for the production of thin sections, for example using a microtome, so that thin, transparent sections having a thickness on the order of micrometers can be produced using a knife. For this purpose, the tissue sample must first pass through a treatment process in which it is fixed, dehydrated, cleared, and then infiltrated with a carrier material, preferably melted paraffin. These processes are often performed successively in a single unit called a “tissue processor”; this tissue processor contains a closable process chamber called a “retort” that receives the various reagents for carrying out the process steps at a suitable temperature and pressure.
One important process step in this context is infiltration of the tissue sample with the carrier material in order to stabilize and consolidate it. This infiltration process step is preceded by the clearing step, in which alcohol residues still present from the preceding dehydration step are removed. The chemical solution used for this clearing step is xylene or a similar medium. In the subsequent infiltration step, in which the tissue sample is exposed to the carrier material (usually melted paraffin), xylene constituents that still remain are flushed out and taken up by the liquid carrier material, with the result that the carrier material in the retort becomes contaminated. Constituents from the tissue sample itself can also contaminate the carrier material. It is therefore necessary to divide the overall infiltration process into multiple individual steps in which the tissue sample is exposed successively to different carrier materials of increasing purity. If the infiltration process is divided into three process steps, for example, the tissue sample is then first treated with a first carrier material that can have a relatively high level of contamination (e.g. with xylene). This is followed by a second infiltration step using a second carrier material that has a higher degree of purity than the first carrier material. Lastly, the tissue sample is exposed to a third carrier material that has the highest degree of purity. In this fashion the tissue sample is completely infiltrated, in a graduated process in which the carrier material for treatment is of increasing purity, with carrier material that has sufficient quality to produce a good thin section in a microtome, and for a microscopic prepared specimen.
The use of multiple liquid carrier materials having different degrees of purity requires that these carrier materials be kept available in containers in a liquid state. If one of the carrier materials is too highly contaminated, this usually affects the aforesaid first carrier material, and it must be replaced with a carrier material having an improved degree of purity. In the case of paraffin, it must be melted from its solid state as stored, typically in the form of paraffin pellets or flakes, until the quantity necessary for replacement is available. Because the carrier material often contains certain chemical additives that are volatile, the melting operation must occur relatively slowly; in practice, this can take several hours. If melting is accomplished in a container inside the tissue processor, said tissue processor is then not operational for that period of time. If the melting process is accomplished outside the tissue processor, this has the disadvantage that the handling of hot, liquid carrier material is relatively difficult for operators; the filling operation into a container inside the tissue process is especially problematic. The bulk volume of paraffin pellets or flakes is considerably greater than the liquid volume of the melted paraffin for the same unit weight. As the paraffin melts it is therefore necessary to top up the melting container several times with solid paraffin in order to obtain the desired volume of molten, liquid paraffin in a compact container.
WO 2006/089365 A1 discloses a method and an apparatus for tissue treatment in which liquid paraffin is used for infiltrating tissue samples. The solid starting material used is blocks of paraffin that are melted in special containers inside a tissue processor. The use of paraffin in block form has the advantage that the desired quantity of liquid paraffin can be estimated relatively accurately based on the size of the block shape.